Sterol glucoside toxins

ABSTRACT

The invention relates to the identification of sterol glucoside toxins, and provides methods for detecting and detoxifying the compounds, as well as therapeutic methods for treating subjects exposed to such toxins. In alternative embodiments, the toxins may for example include beta-sitostrol-beta-D-glucoside (5-cholesten-24b-ethyl-3b-ol-D-glucoside) or cholesterol glucoside (5-cholesten-3b-ol-3b-D-glucoside).

FIELD OF THE INVENTION

The invention relates to the identification of sterol glucoside toxins,and provides methods for detecting and detoxifying the compounds, aswell as therapeutic methods for treating subjects exposed to suchtoxins.

BACKGROUND OF THE INVENTION

Sterols are a diverse group of lipids, many of which are found inappreciable quantities in animal and vegetal tissues. Sterols mayinclude one or more of a variety of molecules belonging to C27-C30crystalline alcohols, having a common general structure based on thecyclopentanoperhydrophenanthrene ring (also called sterane). In thetissues of vertebrates, the main sterol is the C27 alcohol cholesterol.There are a variety of other naturally-occurring animal sterols, such aslanosterol (a C30 compound) and 7-dehydrocholesterol, which areillustrative of the structural similarities of sterols. Thenomenaclature of sterols is based on the numbering of the carbons asexemplified below for cholesterol:

Sterols are also found in plants. The denomination “phytosterol” hasbeen used for sterols of vegetal origin. Chemically, plant sterolsgenerally have the same basic structure as cholesterol, with differencesoccurring for example in the lateral chain on carbon 17. Cholesterol mayitself be found in plants. Representative phytosterols are compoundshaving 29 or 30 carbon atoms, such as campesterol, stigmasterol andbeta-sitosterol (stigmasta-5-en-3beta-ol).

Steryl glycosides are sterol derivatives in which a carbohydrate unit islinked to the hydroxyl group of a sterol molecule. In plants, sterylglycosides have been found in which the sterol moiety is composed ofvarious sterols: campesterol, stigmasterol, sitosterol, brassicasteroland dihydrositosterol. Similarly, the carbohydrate moiety may becomposed of a variety of sugars, such as glucose, xylose or arabinose.Sterol glycosides may be obtained from biological sources such as planttissues by a variety of methods (see for example Sugawara et al. Lipids1999, 34, 1231; Ueno, et al. U.S. Pat. No. 4,235,992 issued Nov. 25,1980). An exemplary plant sterol glycoside isbeta-sitostrol-beta-D-glucoside(5-cholesten-24b-ethyl-3b-ol-D-glucoside), for which the formula is givebelow (also showing the structure of the acylated compound):

Acylated sterol glycosides may be formed in plants when a fatty acid isacylated at the primary alcohol group of the carbohydrate unit (such asglucose or galactose) in the steryl glycoside molecule (see Lepage, JLipid Res 1964, 5, 587). For example, the 6′-palmitoyl-beta-D-glucosideof beta-sitosterol is reportedly present in potato tubers and the6′-linoleoyl-beta-D-glucoside of beta-sitosterol is reportedly found insoybean extracts. Acylated steryl glucoside may be present at relativelyhigh concentrations in a variety of vegetable parts, with the acylatedform being generally more abundant that the non acylated sterolglycoside itself (Sugawara et al., Lipids 1999, 34, 1231).

Sterol glycosides also occur in bacteria. Helicobacter has for examplebeen described as being particularly rich in cholesterol glucosides(Haque et al., J. Bacteriol 1995, 177: 5334; Haque et al., April 1996, JBacteriol;178(7):2065-70). A cholesterol diglucoside has been reportedto occur in Acholeplasma axanthum (Mayberry et al., Biochim Biophys Acta1983, 752, 434).

Sterols and sterol glycosides have been reported to have a wide spectrumof biological activities in animals and humans (Pegel, et al., U.S. Pat.No. 4,254,111 issued Mar. 3, 1981; Pegel et al., U.S. Pat. No. 4,260,603issued Apr. 7, 1981) and techniques for transdermal administration ofthese compounds have been suggested (Walker, et al. U.S. Pat. No.5,128,324 issued Jul. 7, 1992). It has been suggested that some plantsterols, their fatty acid esters and glucosides may be useful fortreating cancers (Eugster, et al., U.S. Pat. No. 5,270,041, Dec. 14,1993). There have been indications that sterols and sterol glycosidesare generally non-toxic, or toxic only at relatively high doses whilebeing beneficial at lower doses (Pegel, U.S. Pat. No. 4,188,379 issuedFeb. 12, 1980). Some phytosterols are thought to have therapeuticeffects, such as anti-tumor properties. Beta-sitosterol is catogorizedin the Merk Index, Tenth Edition, as an antihyperlipoproteinemic. It hasbeen suggested that beta-sitosterol (BSS), and its glucoside (BSSG)enhance the in vitro proliferative response of T-cells (Bouic et al.,Int J Immunopharmacol 1996 December;18(12):693-700), may have otherstimulatory effects as immunomodulators (Bouic et al., Int J Sports Med1999 May;20(4):258-62), and may therefore be therapeutically beneficialin a wide variety of diseases because of these immunostimulatoryproperties (Bouic and Lamprecht, Altern Med Rev 1999 June;4(3):170-7;Bouic et al., U.S. Pat. No. 5,486,510, Jan. 23, 1996).

Cholesterol glucoside (5-cholesten-3b-ol-3b-D-glucoside) is reportedlymade y human cells in culture in conjunction with a heat shock response(Kunimoto et al., an 2000, Cell Stress Chaperones;5(1):3-7). Cholesterylglucoside has also been eported to occur in Candida bogoriensis(Kastelic-Suhadolc, Biochim Biophys Acta 1980 Nov. 7;620(2):322-5).

Sterol glucosides may be hydrolyzed in acid, such as in methanolic HCl(Kastelic-Suhadolc, Biochim Biophys Acta 1980 Nov. 7;620(2):322-5).Enzymatic cleavage of the beta-glycosidic linkage may also beaccomplished, for example by a beta-d-glucosidase. A thermostablebeta-d-glucosidase from Thermoascus aurantiacus that hydrolysed aryl andalkyl beta-d-glucosides has for example recently been reported (Parry etal., 1 Jan. 2001, Biochem J, 353(Pt 1):117-127). Asteryl-beta-glucosidase (EC 3.2.1.104; CAS Registration No. 69494-88-8;cholesteryl-beta-D-glucoside glucohydrolase) has been identified fromSinapis alba seedlings that reportedly acts on glucosides of cholesteroland sitosterol, but not on some related sterols such as coprostanol, tohydrolyse the glucoside—producing sterol and D-glucose (Kalinowska andWojciechowski, 1978, Phytochemistry 17: 1533-1537).

Selective neuronal cell death is the common hallmark of variousneurodegenerative disorders. At least two mechanisms of neuronal deathhave been identified within the mammalian central nervous system:necrosis and apoptosis. Necrosis is generally characterized as a passiveform of ‘accidental’ cell death that follows physical damage and isdistinguished by membrane permeability changes leading to swelling ofcell organelles and rupture of the plasma membrane (Simonian and Coyle,1996). In contrast, apoptosis is generally characterized as an activeform of programmed cell death involving individual cells that oftenremain surrounded by healthy neighbors. Apoptosis reportedly requiresATP and protein synthesis (Earnshaw, 1995) and has been characterized bycell shrinkage, membrane blebbing, and genomic fragmentation (Ellis etal., 1991; Nagata, 1997).

Both necrosis and apoptosis may be induced by stimulation of neurons byglutamate agonists acting through various glutamatergic excitatory aminoacid (EAA) receptor subtypes (Choi, 1995). The actions of glutamate havebeen classified as either “excitotoxicity” or“excitotoxicity-independent”. Excitotoxicity is thought to involve theover-activation of target EAA receptors leading to increased ionic flux.Two main types of excitotoxicity have been described: (1) chronic/slowexcitotoxicity, which is thought to result from defects in energymetabolism leading to persistent receptor activation by ambientglutamate (Zeevalk and Nicklas, 1990); and, (2) acute/fastexcitotoxicity, which is thought to occur following exposure to highlevels of glutamate or glutamate agonists. For example, theover-stimulation of NMDA receptors by glutamate or NMDA may result inincreased calcium flux, which in turn may lead to activation of cellularproteases and the activation of other potentially harmful molecules orpathways. It has been suggested that such actions may underlie thedamage caused by ischaemia and hypoxia (Choi, 1995; Meldrum andGarthwaite, 1990) or head trauma (Katayama et al., 1988).

Excitotoxicity-independent mechanisms of cell death have been shown toarise due to the accumulation of reactive oxygen species (ROS),elevation of calcium, and the loss of intracellular glutathione (GSH)(Tirosh et al., 2000). Each of these events may induce oxidative stress,described as an imbalance between oxidants (ROS) and antioxidants (GSH,GSH peroxidase, vitamins C and E, catalase, SOD, etc.) with the oxidantsbecoming dominant (Sies, 1991). Oxidative stress may trigger cellularnecrosis (Wullner et al., 1999) as well as apoptosis (Zaman and Ratan,1998; Hockenbery et al., 1993; Higuchi and Matsukawa, 1999; Nicole etal. 1998) and often arises due to factors leading to GSH depletion. Fora number of reasons, neurons are thought to be particularly susceptibleto oxidative stress, and oxidative stress-induced cell death has figuredin a number of hypotheses concerning neurodegenerative diseases (seeEvans, 1993; Simonian and Coyle, 1996; Palmer, 1999; Russel et al.,1999) and aging (Verarucci et al., 1999).

Toxins present in the environment may play a role in human pathology.For example, agenized wheat flour was the most common source ofprocessed flour in much of the Western world for the first fifty yearsof the 20^(th) Century (see Shaw and Bains, 1998; Campbell et al., 1950)and was later found to contain methionine sulfoximine (MSO) in highconcentration. MSO induced epileptic seizures in experimental animals((Newell et al., 1947), an action that was not understood but thought toarise due to MSO acting to inhibit the synthesis of both GSH andglutamine (Meister and Tate, 1976). Subsequent studies have revealedthat MSO also has neuro-excitotoxic actions, apparently via NMDAreceptor activation (Shaw et al., 1999).

The etiology of various age-related neurological diseases remainslargely unknown. Sporadic forms of Alzheimer's, Parkinson's, and LouGehrig's disease (amyotrophic lateral sclerosis, ALS) have been linkedto environmental factors that cause neuronal cell death by either byexcitotoxicity or by inducing oxidative stress. The experimental andclinical literature has been taken to support a potential role forexcitotoxins in some forms of neurodegeneration, notably Lou Gehrig'sdisease and Alzheimer's disease. In particular, abnormalities inglutamate handling/transport have been linked to ALS (Rothstein et al.,1990, 1992, 1995) and domoic acid, a kainate receptor agonist, has beenshown to be causal to memory losses much like those reported inAlzheimer's disease (Perl et al., 1990). Oxidative stress has also beenlinked to the same diseases, particularly following GSH depletion (seeBains and Shaw, 1997). Excitotoxicity and oxidative stress may in factbe innately linked in that neural excitation, particularlyover-excitation which occurs in excitoxicity, may generate free radicalsacting to increased oxidative stress (Bindokas et al., 1998).

The following abbreviations may be used in the present application: ALS,amyotrophic lateral sclerosis; ALS-PDC, ALS-parkinsonism dementiacomplex; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;ATP, adenosine triphosphate; BSSG, β-sitosterol-β-D-glucoside; EAA,excitatory amino acid; GluR, glutamate receptor; GSH, glutathione; LDH,lactate dehydrogenase; MSO, methionine sulfoximine; NMDA,N-methyl-D-aspartate; ROS, reactive oxygen species; SOD, superoxidedismutase.

SUMMARY OF THE INVENTION

In one aspect, the present invention discloses the neuronalexcitotoxicicity of sterol glycosides. In alternative embodiments,sterol glycosides that are characterized by neuronal excitotoxicity areβ-sitosterol-β-D-glucoside (BSSG) and cholesterol glucoside.

In one aspect of the invention, BSSG is identified as a toxin present inthe seed of the cycad palm (Cycas circinalis), historically a staple ofthe diet of the Chamorro people of Guam. Cycad seed consumption has beenlinked to ALS-parkinsonism dementia complex (ALS-PDC), an endemicneurological disorder of Guam (Kurland, 1988). Accordingly, in variousembodiments, the present invention provides methods of treating foods toreduce the concentration of sterol glycosides such as BSSG orcholesterol glucoside in foods. In some embodiments, the foods to betreated may for example include plant materials.

An alternative aspect of the present invention is the demonstration thatmice fed cycad flour containing BSSG have severe behavioralabnormalities of motor and cognitive function, as well as significantlevels of neurodegeneration in the cortex, hippocampus, spinal cord,substantia nigra and other CNS regions measured post mortem.Accordingly, in one aspect the present invention provides an animalmodel for studying neurodegenerative disease, in which a non-humanmammal is fed an excitatory neurotixic sterol glycoside such as BSSG orcholesterol glucoside.

In one aspect, the present invention demonstrates that BSSG may mediateneuronal glutamate release followed by NMDA receptor activation.Accordingly, in one aspect the present invention provides in vitroassays for modulators of cytotoxic action, such as assays foridentifying compounds that interfere with cytotoxic neuronal glutamaterelease mediated by BSSG or cholesterol glucoside. Lactate dehydrogenaseassays may for example be used to assay cell death in vitro inconjunction with administration of BSSG and putative inhibitors ofcytotoxicity.

In an alternative aspect, the invention provides kits for detecting BSSGor cholesterol glucoside, for example to detect BSSG or cholesterolglucoside in foods or in body fluids. Such kits may for example includeimmunoassays. In one aspect, the invention accordingly providesantibodies, or other ligands, that bind to the toxins of the invention.

In further alternative aspects, the invention provides methods fortreating subjects exposed to the toxins of the invention. For example,such methods may include vaccination with an antigenic compositioneffective to raise antibodies to the toxins, or treatment of body fluidswith an adsorbent, such as an immunoadsorbent, to remove toxins of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Shows neuro-excitotoxic action of cycad extract (7× washed cycadchips) demonstrated by in vitro indices of cycad-induced neural activityand toxicity on rat cortical slices. A. Cortical wedge recording ofadult rat neocortex. Drugs were administered to the medium bathing eachwedge by gravity flow and neural activity differentially recorded as afield potential. MK801 (MK) blocked the NMDA and cycad-induceddepolarizations as did AP5 (not shown); NBQX blocked only the AMPAresponse, but had no effect on the cycad response (not shown). B.Cortical slice assays for LDH release following exposure to variouscompounds. Cycad fractions in the same concentration as applied toinduce depolarization gave greater LDH release than that evoked by NMDA.The effects of both were attenuated by AP5. Mg²⁺ diminished LDH releasewhile freeze-thawing slices maximized cell death. *P<0.05, Student's ttest.Drug concentrations: NMDA (N), 20 μM; AMPA (A), 10 μM; cycad: 1:50dilution of crude extract of washed cycad in Krebs-Heinseleit buffer(Cyc).

FIG. 2. Actions of isolated BSSG fractions on rat cortical slices. A.Field potential recording of isolated cycad sterol glucoside fractionD-2 (15 μM) compared to NMDA (20 μM), other plant sterol glucosides(ouabain or emicymarin, 50 μM), the β-sitosterol aglycone (10 μM), orD-2 plus AP5 (10 μM). Arrows indicate onset of drug application. B. LDHrelease following exposure to the same BSSG D-2 fraction (75 μM)compared to NMDA (50 μM), the sitosterol aglycone, β-SS (75 μM); variouscompounds in the presence of AP5 (20 μM). The action of cholesterolglucoside was qualitatively similar to β-sitosterol-β-D-glucoside (notshown). Statistics as in FIG. 1.

FIG. 3: [³H]-glutamate release in rat cortical slices. A. [³H]-glutamaterelease with isolated BSSG D-1-2 fraction (25 μM) compared to NMDA (50μM) with or without AP5 (20 μM). For this experiment, calciumconcentration was either 0 (L) or 2 mM (H). Note the calcium dependencefor both NMDA and BSSG. B. [³H]-glutamate release by D-2 BSSG fraction.Concentrations as in B.

FIG. 4: Behavioral test results in the mouse model of neurodegenerativedisease. A. Leg extension: the mouse is held by its tail, and in anormal mouse, both of its legs flex out (a score of 2 is recorded). Ifone or both of the legs do not flex out a score of 1 or 0 is givenaccordingly. B. Gait Length: the mouse walks through a tunnel with painton its backpaws. Distance between subsequent paw prints is recorded asthe gait length (stride length). C. Rotarod: the mouse is placed in arotating cylinder, at increasing speeds. The time to fall of thecylinder and number of spins (rotations with out falling off) arerecorded. D. Wire Hang: the mouse is placed up side down on a wire meshand time to fall into a padded box is recorded. E. Water Maze: the mouseis placed in a small swimming pool of water and swims to find a hiddenplatform located near the middle of the pool. Time to find the platformand percentage of time in each quadrant of the pool is recorded. F.Radial Arm Maze: the mouse is placed in a 8 arm maze, in which 4 of thearms are baited with food. Errors are recorded as entries into unbaitedtubes and re-entry in to tubes already visited.

FIG. 5: Caspase-3 labeling of human cortical astrocytes in tissueculture. Fetal human telencephalic astrocytes were grown to confluencyand then exposed to NMDA, BSSG, cholesterol glucoside (CG), or hydrogenperoxide (H₂O₂) for various periods. The peak of caspase-3 positivelabeling was seen at 24 hrs. after exposure to the various compounds.Note significant caspase-3 positive labeling in all experimentalconditions vs. control (DMEM), including for the sterol glucosides BSSGand CG. At longer time points, the overall numbers of cells in theseconditions declined.

FIG. 6: Cumulative motor and cognitive deficits of seven cycad-fedanimals (cyc 1 through cyc 7). Averages of the values recorded for eachof the cycad animals on each of the behavioural tests described wereexpressed as a percentage in relation to the cumulative control values.Values were computed for motor, cognitive, or combined motor andcognitive functions as follows: Control values for any measure wereaveraged across all control animals and set to 100%. For each cycadanimal, the individual response for each separate measure was describedas % response/100; total cumulative response for cycad mouse is the sumaverage of all such separate measures/100. Left: Motor deficits. Centre:Cognitive deficits. Right: Combined motor and cognitive deficits. S.E.M.indicates variance across individual behavioural measures in each of thecycad-fed mice.

DETAILED DESCRIPTION OF THE INVENTION

Kits and assays of the invention may include a variety of techniques fordetecting toxins. For example, antibodies to BSSG or cholesterolglucoside may be used in kits or assays. Antibodies, or other ligands,that bind to the toxins of the invention may for example be used toprepare kits such as immunoassay agglutination kits designed to detectthe toxins in biological specimens, such as blood or feces. Affinitypurified antibodies against toxins of the invention may for example beused to passively coat small particles, such as polystyrene particles,that form visible aggregates when they are mixed with a samplecontaining the toxins. Many alternative immunoassay procedures fordetecting toxins of the invention in body fluids may be adapted frommethods known in the immunoassay art. Such techniques may includeradio-immunoassay techniques; and, enzyme-immunoassay techniques such ascompetitive, double antibody solid phase (“DASP”) and sandwichprocedures. Various solid phase immunoassays may be performed usingvarious solid supports, including finely divided cellulose, solid beadsor discs, polystyrene tubes and microtiter plates. Enzyme immunoassaysmay be adapted to include color formation as an indicator of a result.

Procedures for raising polyclonal antibodies are also well known.Typically, such antibodies can be raised by administering an antigenicformulation of the toxin of the present invention subcutaneously to anantibody producing animal, such as New Zealand white rabbits. Theantigens may for example be injected at a total volume of 100microlitres per site at six different sites. Each injected material maycontain adjuvants. The rabbits are then bled two weeks after the firstinjection and periodically boosted with the same antigen three timesevery six weeks. A sample of serum is then collected 10 days after eachboost. Polyclonal antibodies are then recovered from the serum byaffinity chromatography using the corresponding antigen to capture theantibody. This and other procedures for raising polyclonal antibodiesare disclosed in E. Harlow, et. al., editors, Antibodies: A LaboratoryManual (1988), which is hereby incorporated by reference. U.S. Pat. No.5,753,260 issued to Alving, et al. May 19, 1998 (incorporated herein byreference) discloses immunoreactive compositions and methods forimmunizing animals to produce antibodies against sterols, which may beused to produce anti-BSSG or anti-cholesterol glucoside antibodies.Antigenic compositions for raising such antibodies may for exampleinclude liposomes containing phosphatidylcholine, toxin (such as BSSG orcholesterol glucoside), and adjuvant such as lipid A in molar ratios ofapproximately 2:5:0.02. Delivery vehicles other than liposomes wouldalso be suitable, including microcapsules, microspheres, lipospheres,polymers, and slow release devices could serve instead of liposomes.

Monoclonal antibody production to toxins of the present invention maysimilarly be effected by known techniques involving first obtainingimmune cells (lymphocytes) from the spleen of a mammal (e.g., mouse)which has been previously immunized with the antigen of interest eitherin vivo or in vitro. The antibody-secreting lymphocytes are then fusedwith (mouse) myeloma cells or transformed cells, which are capable ofreplicating indefinitely in cell culture, thereby producing an immortal,immunoglobulin-secreting cell line. Fusion with mammalian myeloma cellsor other fusion partners capable of replicating indefinitely in cellculture is effected by standard and well-known techniques, for example,by using polyethylene glycol (“PEG”) or other fusing agents (seeMilstein and Kohler, Eur. J. Immunol. 6:511 (1976), which is herebyincorporated by reference). The resulting fused cells, or hybridomas,are cultured, and the resulting colonies screened for the production ofthe desired monoclonal antibodies. Colonies producing such antibodiesare cloned and grown either in vivo or in vitro to produce largequantities of antibody. A description of the theoretical basis andpractical methodology of fusing such cells is set forth in Kohler andMilstein, Nature 256:495 (1975), which is hereby incorporated byreference.

In addition to utilizing whole antibodies, the kits and processes of thepresent invention encompass use of binding portions of such antibodiesthat recognize toxins of the invention. Such binding portions includeFab fragments, F(ab′)₂ fragments, and Fv fragments. These antibodyfragments can be made by conventional procedures, such as proteolyticfragmentation procedures, as described in Goding, Monoclonal Antibodies:Principles and Practice, pp. 98-118, New York: Academic Press (1983),which is hereby incorporated by reference.

In one aspect of the invention, ligands (such as antibodies) to thetoxins of the invention may be administered to a patient in need of suchtreatment to ameliorate the effect of the toxins on the patient. In analternative aspect of the invention, an animal, such as a human, may bevaccinated with an antigenic composition effective to raise antibodiesagainst sterol glucoside toxins of the invention, such as BSSG orcholesterol glucoside, to retard or reduce the severity of toxicitycaused by ingestion of toxins of the invention.

In alternative embodiments, the invention provides methods ofdetoxification of compositions containing toxins, such as foodstuffs, inwhich sterol glucoside toxins of the invention may be hydrolyzed, forexample in acid, such as in methanolic HCl (Kastelic-Suhadolc, BiochimBiophys Acta 1980 Nov. 7;620(2):322-5). Detoxification by enzymaticcleavage of the beta-glycosidic linkage may also be accomplished, forexample by a beta-d-glucosidase. A thermostable beta-d-glucosidase fromThermoascus aurantiacus that hydrolyses aryl and alkyl beta-d-glucosideshas for example recently been reported (Parry et al., 1 Jan. 2001,Biochem J, 353(Pt 1):117-127). A steryl-beta-glucosidase (EC 3.2.1.104;CAS Registration No. 69494-88-8; cholesteryl-beta-D-glucosideglucohydrolase) has been identified from Sinapis alba seedlings thatreportedly acts on glucosides of cholesterol and sitosterol (Kalinowskaand Wojciechowski, 1978, Phytochemistry 17: 1533-1537).

In one aspect, the invention provides methods for treatment ofmaterials, such as body fluids, with an adsorbent, such as animmunoadsorbent, to remove toxins of the invention. In such methods, anantibody in an insoluble form may be used to bind the toxin antigen toremove it from a mixture of substances. For example, solid-phaseimmunoadsorbent gels may be used, in which purified antibodies, forexample from the serum of immunized animals, is coupled to cyanogenbromide-activated 4% agarose gels. In alternative embodiments, sephadex,derivatives of cellulose, or other polymers can be used as the matrix asan alternative to agarose.

Alternative aspects of the invention are illustrated in the followingExamples, which are merely illustrative of some embodiments and do notnecessarily reflect the full scope of the invention.

EXAMPLES

Animals

In vitro experiments were performed on adult (>70 do) maleSprague-Dawley colony rats maintained on a light-dark cycle (12 hr:12hr). In vivo experiments were conduced using CD-1 colony reared 5-7 mo.old male mice.

Chemicals

MSO was obtained from Sigma-Aldrich Canada Ltd. (Mississauga, Ontario).AMPA, NMDA, AP5, and NBQX were obtained from Precision Biochemicals Inc.(Vancouver, British Columbia). [³H] CGP 39653 and [³H] glutamate werepurchased from NEN/Mendel Scientific Co. (Guelph, Ontario). LDH kits andDNAase were obtained from Sigma (St. Louis). TUNEL kits were purchasedfrom Intergen (ApopTag). (Oxford). Other chemicals were of analyticalgrade available from BDH Inc. (Vancouver, British Columbia).

Cycad Extracts and Purification of BSSG

Initial experiments were performed with crude cycad flour extracts madeby extensively grinding chips of cycad in a small volume of distilledwater. These cycad chips had been extensively soaked over a period of 7days. This cycad extract was diluted by various factors inKrebs-Henseleit buffer for use in bath application to field potential orLDH assays. Based on early experiments (e.g., see FIG. 2), cycadfractions were extensively screened for potency based on the size of theevoked field potential response or on amount of LDH released. From eachstage, the most potent batch was selected and further separated bycolumn chromatography. The fractions ultimately yielded several variantsof a sterol glucoside, β-sitosterol-β-D glucoside (BSSG) with a range ofmolecular weights ranging from 574-576). These fractions have been givenfraction identification codes indicating stage in the isolationprocedure and are described in the following as D-2, D-1-1, and D-2.

Electrophysiology: Field Potential Recordings

Cortical ‘wedges’ were prepared as described previously (Shaw et al.,1996). In brief, animals were anesthetized with CO₂, decapitated, and acortical block rapidly removed and placed in cold Krebs-Henseleit buffercontaining (in mM): NaCl 124, KCl 3.3, NaHCO₃ 25, glucose 10, KH₂PO₄1.2, CaCl₂ 2.4, and MgSO₄ 1.2, bubbled with 5% CO₂/95% O₂, pH 7.4. Thecortical block was sectioned into 500 μM thick coronal slices using aVibratome (Campden Instruments) and the slices cut into pie shapedwedges in which the white matter formed the narrow edge of the wedge.Each wedge was placed on a net across a grease gap between two fluidfilled chambers. The cortical side of the wedge was bathed (at roomtemperature, approx. 25° C.) in buffer lacking Mg²⁺; the callosalportion was bathed in buffer containing Mg²⁺ to minimize neuralactivity. Field potentials were differentially recorded between the twochambers using two Ag/AgCl electrodes. Recordings from up to 6 wedges,each in individual chambers, could be made simultaneously for eachexperiment. The wedges were continuously perfused on the cortical sidewith oxygenated, Mg²⁺-free buffer using a gravity feed system. Usingthis system, drugs could be rapidly substituted for control media toexamine response characteristics. Wedges typically survived for up to 8hrs. Responses were recorded on LabView™ after amplification and A/Dconversion and the traces were charted in Excel™ for Windows™.Statistical analysis of peak response amplitude was performed by one-wayANOVA using Bonferroni's post test with GraphPad Prism™.

[³H]-Glutamate Release Studies

Brain slices were taken from cortical blocks in which all subcorticaltissue had been removed. 400 μM slices were cut using a modified slicecutter (Van Huizen et al., 1989). Slices were rinsed twice for 5 min inMg⁺² containing Krebs-Henseleit buffer pH 7.4. Incubation mediaconsisted of 100 μM cold glutamate, 20 μM AP5 and 10 μM DNQX, the latterNMDA or AMPA antagonists, respectively. 10 nM of [³H]-glutamate wasadded to the mixture and incubated for 1 hr at 37° C. under inoxygenated atmosphere (O₂/CO₂=95/5%). Experimental treatments wereperformed in 500 μM Mg⁺² free buffer placed in tissue culture wellscontaining different concentrations of MSO or isolated BSSG fractions ofcycad flour. Slices were removed at the end of incubation period and thesupernatant removed for scintillation counting. The supernatantfractions were placed in scintillation vials containing NEN Formula 989for a minimum of 12 hrs before being counted in a Beckman LS6000scintillation counter. Results were normalized to the dpm counts ofrespective controls.

LDH Assays

Cortical slices were prepared as described above in the glutamaterelease experiments and placed in tissue culture wells containingKrebs-Heinsleight buffer supplemented with 0.0004% H₂O₂ and 1 mg/mLglucose. Extensive previous studies have demonstrated that this mediumsupports cellular activity for prolonged periods (Van Huizen et al.,1989; Shaw et al., 1996). (Note that hydrogen peroxide, added as thesource of molecular oxygen, was not deleterious at this lowconcentration (see Van Huizen et al., 1989). In our preliminaryexperiments, hydrogen peroxide did not affect LDH release up to a 1 mMconcentration (0.0034%). All slices were washed twice with buffer for 20min each at room temperature before incubation in media containing thetest compounds for 1 hr at 37° C. Test compounds included MSO, NMDA,kainate, different concentrations of cycad extract or differentfractions or concentrations of isolated BSSG. MSO, NMDA, and cycad/BSSGwere each tested alone or in combination with AP5, and compared tocontrol slices maintained in buffer alone. For additional comparison andto establish the limits of the method, some slices were freeze-thawed tokill all the cells. Alternatively, some slices were incubated in buffercontaining 1.2 mM Mg²⁺ in order to diminish spontaneous neural activity.At the end of the 1 hr incubation period, 3 samples (100 μl of buffer,each sample) were taken from each well. LDH assays were performed onthese samples using a LDH diagnostic kit (Sigma) following themanufacturer's protocol with some modifications. In brief, 0.5 ml ofpyruvate solution was mixed with 0.5 mg pre-weighed NADH. 100 μl ofslice medium (free of slices) was added to the mixture and incubated for30 min at 37° C. 0.5 ml of Sigma coloring reagent (2,4-dinitrophenylhydrazine in HCl, 2 mg/ml) was added to develop the colorand the mixture was incubated for 20 min at room temperature. 5 ml of0.4 N NaOH were added to each tube. After 5 min, optical density wasread at 440 nm. Standard curves were prepared for each assay usingdifferent concentrations of pyruvate solution (0-960 units). LDHactivity (in International Units) was calculated from the standard curveand normalized by total protein content of each slice as determined bythe Lowry protein assay (Peterson, 1979). One International Unitrepresents the amount of enzyme required to convert 1 μmol ofsubstrate/minute at room temperature.

In Situ Labeling of DNA FRAGMENTATION/Apoptosis

Terminal deoxynucleotidyl transferase (TdT) mediated dUTP-digoxigenin(DIG) nickend labeling (TUNEL) was carried out using an Intergen ApopTagPlus peroxidase kit using the manufacturer's protocol adapted fromGavrieli et al. (1992) with some modifications. More specific antibodylabels for apoptosis, eg. caspase 3 also showed cell death in the sameregions. Briefly, 20 μM thick coronal sections were cut on a cryostatthen fixed in 1% paraformaldehyde at room temperature for 2 days. Theendogenous peroxidase was quenched by 3% hydrogen peroxide in phosphatebuffered solution (PBS). After rinsing with PBS, the sections were thenexposed to 11 μL/cm² working strength of TdT enzyme for 1 hr at 37° C.After washing in PBS, 15∥L/cm² of anti-digoxigenin-peroxidase wasapplied for 30 min in a humidified chamber at room temperature. Colourwas developed by adding 125 μl DAB substrate working solution for 6 min.Slides were counter-stained with methyl green for 25 minutes at roomtemperature. Positive apoptosis controls were generated bypre-incubating sections with DNAase (Sigma). These methods have beensuccessfully used to indicate apoptotic neurons when used in otherpreparations (Simpson et al., 2000).

Data for LDH and glutamate release experiements were analyzed forsignificance by one way ANOVA using Dunnett's and Bonferroni's posttests with GraphPad Prism™.

Tissue Culture Studies of BSSG Toxicity

We have raised cortical astrocytes on coverslips in culture, exposingthem to kainic acid, BSSH, or cholesterol glucoside. Cell loss wasmeasured directly by cell density measurements; apoptosis was measuredby staining cells for caspase-3 labeling. Each of these compoundsgenerated a time-dependent apoptotic cell loss. These data are shown inFIG. 5.

Results

MSO and cycad Mechanisms of Action in CNS

MSO, crude cycad extract, and BSSG isolated from cycad seed flour weretested for neural action and neuro-excitotoxicity in a series ofbioassays. FIG. 1 a shows the neural response to MSO measured as fieldpotential in the cortical wedge preparation from adult rat. Bathapplication of MSO led to a relatively rapid depolarizing fieldpotentials over a range of concentrations beginning at approx. 50 μM.The responses to glutamate receptor agonists NMDA and AMPA are alsoshown in the traces of FIG. 1 a for comparison. MSO responses, likethose of NMDA, could be blocked by the co-application of NMDA receptorantagonists AP5, kynurenate, or MK 801 (the latter not shown here). MSOresponses were not blocked by application of AMPA antagonists NBQX orother AMPA antagonists. FIG. 1 b shows LDH assays for rat corticalslices following exposure to MSO and other excitotoxins. Both NMDA andMSO increased cell death as measured by LDH release, and both treatmentswere blocked by the addition of AP5.

The actions of cycad flour extracts on the cortical wedge preparationare shown in FIG. 2. Cycad extracts gave depolarizing field potentialsthat could be blocked by MK801 (FIG. 2 a) or AP5 (data not shown), butnot NBQX (data not shown). LDH assays confirmed that cycad extractβ-sitosterol-p-D-glucoside fractions gave increased LDH release thatcould induce cell death, an effect that was blocked by AP5 (FIG. 2 b).Assays were also performed for comparison using other plant sterolglucosides (ouabain and emicymarin), and for synthetic cholesterolglucoside. The β-sitosterol agylcone and cholesterol were screened forcomparison to the glucosides. Ouabain and emicymarin gave smallhyperpolarizing responses and gave little LDH release (not shown). Theaglycone sterols were without effect. The action of cholesterolglucoside was qualitatively similar to β-sitosterol-β-D-glucoside (notshown).

FIG. 3 shows results from the cortical wedge preparation (FIG. 3 a) andin LDH assays (FIG. 3 b) using isolated BSSG. The isolated BSSGfractions gave similar field potential responses that were blocked byNMDA antagonists. Cell death in LDH assays was also blocked by NMDAantagonists.

To test whether the actions of MSO and BSSG might act indirectly byreleasing glutamate from intracellular compartments, we examinedradiolabeled glutamate release from rat cortical slices. Preloaded[³H]-glutamate release was significantly increased in the presence ofMSO and BSSG fractions D-1-2 and D-2 (FIG. 4 abc) in a calcium dependentmanner, and these effects could be blocked by AP5.

Cycad-fed animals showed significant and progressive deficits in bothmotor and cognitive function. Post-sacrifice histological examinationsof the brains of cycad-fed animals revealed the presence of significantlevels of apoptosis in hippocampal formation, cortex, and spinal cordcompared to control mice. Rats fed MSO also showed evidence of apoptosisin CNS.

Synthesis of β-sitosterol-β-D-glucoside and Related Analogues.

The above experiments have demonstrated in vitro and in vivoneurotoxicity of cycad and have suggested that the toxic component incycad is β-sitosterol-β-D-glucoside and related sterol glucosides. Totest the hypothesis that P-sitosterol-β-D-glucoside is the active toxinrequires either more of this compound extracted from cycad or thesynthesis of the molecule de novo. As cycad is limited in supply, not tomention tedious to process, we have attempted to provide a syntheticpathway to acquire sufficient P-sitosterol-β-D-glucoside for futureexperiments. We have now accomplished this using the high yield methodsset out below (provided by Drs. D. E. Williams and L. Lermer, UBC).

a. Preparation of D-glucose-pentaacetate

Glucose (20 g, 0.111 mols) and NaOAC (9.10 g, 0.111 mols) is added to adry 1 L RBF under N₂. Acetic anhydride (125.7 ml, 1.332 mol, 12 eq) isadded using a syringe. The solution is rapidly stirred and then warmedusing a Bunsen burner until the solution becomes clear and colorless.The solution is allowed to cool to room temperature, forming a whiteprecipitate. The solution is stirred for 2 hrs, taken up in EtOAc,washed with H₂O 3×, 5% NaHCO₃ 3×, and 1× with brine. The organic layeris dried with MgSO₄, filtered and concentrated under reduced pressure,to afford D-glucose-pentaacetate, a white powder. The powder isre-crystallized in EthOH (approx. 400 ml) to afford 35.96 g of a whitepowder. The re-crystalized D-glucose-pentaacetate gives 42.02 g (65%yield). The mother liquor is concentrated and re-crystallized to afforda second crop of 6.60 g of D-glucose-pentaacetate as a white powder. Theremaining mother liquor is concentrated to afford 18.82 grams of a whitesolid. An over all yield of 92.5% is obtained.

b. Preparation of 2,3,4,6-Tetra-O-acetyl-D-glucopyranose

Hydrazine acetate 5.1 g (55.39 mmol, 1.20 eq) is placed in a dry 500 mlRBF under nitrogen. Dry DMF (dried over CaSO4 and distilled at 5 mmHg)is added to the flask using a syringe. D-glucose-pentaacetate 17.96 g(46.00 mmol) is placed in a dry 250 ml RBF under nitrogen and dissolvedin 100 ml dry DMF. The D-glucose-pentaacetate is added to the reactionflask via a cannula. After the addition of D-glucose-pentaacetate, thesolution has a slightly yellow colour. Solid hydrazine remainedsuspended. After 3 hrs the suspension has dissolved and the solutionremains clear pale yellow in colour. The solution is taken up in EtOAc,washed 3× with H₂O and 1× with brine, dried over MgSO₄, filtered andconcentrated under reduced pressure.2,3,4,6-Tetra-O-acetyl-D-glucopyranose as clear colourless viscous oilis obtained. This oil is used without further purification in the nextstep.

c. Preparation of 2,3,4,6-Tetra-o-acetyl-α-D-glucosyltrichloroacetimidate

The viscous 2,3,4,6-Tetra-O-acetyl-D-glucopyranose oil is dissolved indry CH₂Cl₂ 150 ml in a 500 ml RBF under nitrogen and cooled to −40° C.(cooling bath of CH₃CN/CO₂). Cl₃CCN 46.11 ml (459.9 mmol, 10 eq) isadded to the reaction flask drop wise using a syringe, followed by theaddition of DBU (0.68 ml, 0.1 eq). After two hrs the solution is allowedto warm to room temperature. The solvent is removed under reducepressure and the oil is loaded using a minimum of CH₂Cl₂ on a 8 cmdiameter silica column. The column is eluted with 3:1 pet. ether:EtOACfollowed by 2:1 pet.Ether: EtOAc. Three fractions are collected. Thefirst fraction RF=0.63 contains 16.394 g of2,3,4,6-Tetra-o-acetyl-α-D-glucosyl trichloroacetimidate (α-TAG-I) as aviscous oil. A second oil fraction is collected containing a mixture ofα-TAG-I and small impurity of β-TAG-I 1.644 grams and a third fractioncontaining a 4.060 grams of β-TAG-I as a white powder. A 71% yield of2,3,4,6-Tetra-o-acetyl-α-D-glucosyl trichloroacetimidate is obtained anda total overall yield of 87% is achieved.

d. Coupling—preparation of2,3,4,6-Tetra-o-acetyl-β-D-glucosyl-β-sitosterol

2,3,4,6-Tetra-o-acetyl-α-D-glucosyl trichloroacetimidate (α-TAG-I)(1.771 g, 0.54 mmol, 1.5 eq) is pumped on over night and dissolved indry CH₂Cl₂. The solvent is then removed under low pressure. Dry α-TAG-Iin a 10 ml flask is re-dissolved in 5 ml of CH₂Cl₂. The α-TAG-I istransferred into the 25 ml RBF reaction flask containing activated 3 Åmolecular sieves via a cannula. The solution is stirred for 1 hr overthe sieves to take up any residual water. β-sitosterol (0.0994 g, 0.24mmol) is dissolved in 5 m 1 dry CH₂Cl₂ and transferred in the reactionflask via a cannula. The flask containing sitosterol is rinsed with 1 mlCH₂Cl₂. The rinse is added to the reaction flask. The reaction is cooledto −23° C. (cooling bath of CCl₄/CO₂) and some precipitation occurs. Asyringe is used to inject a 0.99 ml volume of a stock solution of 0.1 mlBF₃.Et₂O in 8 ml CH₂Cl₂ drop wise over 20 min to the reaction flask. Thesolution remains clear and colorless. After 2 hrs no precipitationremains in the reaction flask. After 4 hours a fine white precipitate isobserved. The reaction is completed by TLC using (9:1 CHCl₃:MeOH). Asecond addition of TAG-I (0.1180 g, 0.239 mmol, 1 eq) dissolved in 5 mlCH₂Cl₂ is added drop wise to the flask. After 1 hr, no starting materialis present. The solvent is evaporated under reduced pressure and the oilis loaded on a silica column. The column is eluted with 10:1 pet.ether:EtOAc (10 collection tubes) followed by 3:1 pet. ether:EtOAc(15:tube) followed by 1:1 3:1 pet. ether:EtOAc. Two fractions areobtained. The first fraction contains 0.0219 g-β-sitosteryl acetate. Thesecond fraction contains 0.1392 g (78%) of2,3,4,6-Tetra-o-acetyl-β-D-glucosyl-β-sitosterol. (Note: The sterol andthe product have very similar RF's using Pet. Ether: EthOAc solvent toelute. The reaction may be followed using a 9:1 solution of CHCl₃: MeOHto elute the TLCs).

e. Deacetylation—preparation of β-Sitosteryl-β-D-glucopyranoside

The 2,3,4,6-Tetra-o-acetyl-β-D-glucosyl-β-sitosterol 0.1392 g isdissolved in 80 ml warm MeOH. The solution is allowed to cool back toroom temperature. Some precipitation is observed. Et₃N (12 ml) is addedfollowed by 2 ml of H₂O. After three hrs the precipitate hasre-dissolved, and the clear, colorless solution is stirred overnight. Afine white precipitate is observed in the reaction flask the nextmorning. TLC using 15% MeOH/CHCl₃ indicates that the reaction iscomplete. The solvent is removed under reduced pressure to afford awhite solid. The solid is re-dissolved in a minimum amount of 15%MeOH/CHCl₃ The solution is loaded on a silica column eluted with 15%MeOH/CHCl₃. A single white powder 0.1079 g (100% yield) is obtained.These methods result in an overall yield of 78% for the combination ofthe coupling and deacetylation reactions. The white solid isre-crystallized in Ethanol to afford 0.0452 g (42%) of a white powder.The mother liquor is concentrated and a second crop is obtained of0.0165 g (15%) of a white powder. The mother liquor is concentrated andthe solid is re-crystallized using a H₂O/MeOH mixed solvent system toafford 0.0044 g (4%). The remaining mother liquor is concentrated toafford 0.0180 g (17%). The overall yield from the 0.0994 g of sitosterolis 0.0661 g (48%) of re-crystallized β-sitosteryl-β-D-glucopyranosideand 0.018 g (13%) of remaining non-recrystallizedβ-sitosteryl-β-D-glucopyranoside product. Note that the 0.0219 g ofβ-sitosteryl acetate from the coupling reaction can be recycled back tositosterol and then carried through to the desired product in order toincrease yields.

Wedge recording and LDH assays using syntheticβ-sitosterol-β-D-glucoside created by these methods have been shownqualitative similarity between the synthetic and natural D-2 fractions

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CONCLUSION

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. Given the overlap in theoccurrence of particular sterols in plants, animals and other organisms,the present application refers to all such compounds collectively assterols. Numeric ranges are inclusive of the numbers defining the range.In the specification, the word “comprising” is used as an open-endedterm, substantially equivalent to the phrase “including, but not limitedto”, and the word “comprises” has a corresponding meaning. Citation ofreferences herein shall not be construed as an admission that suchreferences are prior art to the present invention. All publications,including but not limited to patents and patent applications, cited inthis specification are incorporated herein by reference as if eachindividual publication were specifically and individually indicated tobe incorporated by reference herein and as though fully set forthherein. The invention includes all embodiments and variationssubstantially as hereinbefore described and with reference to theexamples and drawings.

1. A method of detoxification of a composition, comprising subjectingthe composition to conditions that reduce the concentration of aneurotoxic sterol glycoside in the composition.
 2. The method of claim1, wherein the neurotoxic sterol glycoside is selected from the groupconsisting of beta-sitosterol-beta-D-glucoside and cholesterolglucoside.
 3. The method of claim 1, wherein the sterol glycoside ischolesterol glucoside.
 4. The method of claim 1, wherein the sterolglycoside is beta-sitosterol-beta-D-glucoside.
 5. The method of claim 1,wherein the conditions are effective to hydrolyse a glycosidic bond inthe sterol glycoside.
 6. The method of claim 1, wherein the conditionscomprise treating the composition with an enzyme that degrades theneurotoxic sterol glycoside. 7.-10. (canceled)
 11. A method ofimmunizing an animal comprising administering to the animal an antigenicformulation of a neurotoxic sterol glycoside.
 12. The method of claim11, wherein the neurotoxic sterol glycoside is selected from the groupconsisting of beta-sitosterol-beta-D-glucoside and cholesterolglucoside.
 13. The method of claim 11, wherein the sterol glycoside ischolesterol glucoside.
 14. The method of claim 11, wherein the sterolglycoside is beta-sitosterol-beta-D-glucoside. 15.-18. (canceled)
 19. Akit for detecting neurotoxic sterol glycosides comprising a ligand thatbinds to the neurotoxic sterol glycoside and means for detecting thebinding of the ligand to the neurotoxic sterol glycoside.
 20. The kit ofclaim 19, wherein the ligand is an antibody.
 21. The kit of claim 19,wherein the neurotoxic sterol glycoside is selected from the groupconsisting of beta-sitosterol-beta-D-glucoside and cholesterolglucoside.
 22. The kit of claim 19, wherein the sterol glycoside ischolesterol glucoside.
 23. The kit of claim 19, wherein the sterolglycoside is beta-sitosterol-beta-D-glucoside.
 24. A method foridentifying neurological degeneration in a non-human animal, the methodcomprising the steps of: administering a synthetic neurotoxic sterolglycoside to the animal; and identifying neurological degeneration inthe animal.
 25. The method of claim 24, wherein a glycosidic bond in thesterol glycoside has been hydrolyzed prior to the administering step.26. The method of claim 24 further comprising the step of treating thesterol glycoside with an enzyme that degrades the neurotoxic sterolglycoside.
 27. The method of claim 24 further comprising the step ofdetecting the sterol glycoside in a body fluid or a tissue of theanimal.
 28. The method of claim 27 wherein the sterol glycoside isdetected with an antibody.
 29. The method of claim 27 wherein the sterolglycoside is detected by isolating the sterol glycoside by columnchromatography.
 30. The method of claim 29 wherein the chromatographicmethod is high performance liquid chromatography.
 31. The method ofclaim 24 wherein the neurological degeneration is identified by abehavioral test selected from the group consisting of a leg extensiontest, a gait length test, a rotarod test, a wire hang test, a water mazetest, and a radial arm maze test.
 32. The method of claim 1 wherein theneurotoxic sterol glucoside is selected from the group consisting ofbeta-sitosterol-beta-D-glucoside and cholesterol glucoside.
 33. Themethod of claim 24, wherein the sterol glucoside is cholesterolglucoside.
 34. The method of claim 24, wherein the sterol glucoside isbeta-sitosterol-beta-D-glucoside.
 35. The method of claim 29, whereinthe sterol glucoside is beta-sitosterol-beta-D-glucoside.
 36. The methodof claim 29, wherein the sterol glucoside is cholesterol glucoside.